Deubiquitinase USP19 extends the residual enzymatic activity of phenylalanine hydroxylase variants

Phenylalanine hydroxylase (PAH) is a key enzyme in mammals that maintains the phenylalanine (Phe) concentration at an appropriate physiological level. Some genetic mutations in the PAH gene lead to destabilization of the PAH enzyme, leading to phenylketonuria (PKU). Destabilized PAH variants can have a certain amount of residual enzymatic activity that is sufficient for metabolism of Phe. However, accelerated degradation of those variants can lead to insufficient amounts of cellular PAH protein. The optimal protein level of PAH in cells is regulated by a balancing act between E3 ligases and deubiquitinating enzymes (DUBs). In this work, we analyzed the protein expression and stability of two PKU-linked PAH protein variants, R241C and R243Q, prevalent in the Asian population. We found that the tested PAH variants were highly ubiquitinated and thus targeted for rapid protein degradation. We demonstrated that USP19, a DUB that interacts with both PAH variants, plays a regulatory role by extending their half-lives. The deubiquitinating activity of USP19 prevents protein degradation and increases the abundance of both PAH protein variants. Thus, our study reveals a novel mechanism by which deubiquitinating activity of USP19 extends the residual enzymatic activity of PAH variants.


Results
Generation and analysis of PAH protein mutants. Several mutations in the PAH gene have been studied, and most of them are missense mutations that are spread throughout the PAH protein. The R241C and R243Q mutations, which occur frequently in the Asian population, are considered in this study. The nucleotide aberrations c.721C>T and c.728 G>A in PAH exon 7 code for the p.Arg241Cys (p.R241C) and p.Arg243Gln (R243Q) mutations and are some of the most abundant mutations among PKU patients, with an average allele frequency of approximately 6% (9-14% in Mediterranean countries and the Middle East), with ~ 2% of patients homozygous for this mutation (up to 12% in Mediterranean countries and the Middle East) 9,21 .The PAH protein exists in two distinct conformations 22,23 , an auto-inhibited PAH illustrated in Fig. 1a and an activated PAH conformation (not shown). The auto-inhibited tetrameric structure of human PAH (hPAH) in complex with cofactor BH 4 and the mutation sites (R241C and R243Q) located in the PAH catalytic domain are shown in Fig. 1a.
To generate the c.721C>T (R241C) and c.728 G>A (R243Q) mutations in PAHwt, we used site-directed mutagenesis. The oligonucleotides used to generate the PAH variants are described in Supplementary Table S1. The mutant clones were subjected to Sanger sequencing to confirm the point mutation. The sequencing results confirmed nucleotide aberrations at position 721, C>T, and position 728, G>A (Fig. 1b). The amino acid arginine was converted to cysteine at position 241 (R241C), and arginine was converted to glutamic acid at position 243 (R243Q) (Fig. 1c). We next performed an expression analysis of the two mutant proteins and the PAHwt protein.
We transfected HEK293 cells with constant amounts of HA-tagged PAHwt, R241C, and R243Q and analyzed their expression levels by Western blotting. The expression of PAH was normalized to that of GAPDH. We found that both the R241C and R243Q variants were expressed at lower levels than PAHwt, particularly the R243Q variant, which showed a significant reduction compared with both R241C and PAHwt (Fig. 1d). These point variants have been reported to have residual enzymatic activity 24,25 (https:// www. biopku. org). Figure 1e represents  www.nature.com/scientificreports/ mutation in the PAH gene, residual in vitro enzyme activity, protein stability prediction (FoldX), and assignment to metabolic phenotypes of the PAH gene variations investigated in this study.

PAH variants undergo proteasomal degradation.
The proteasomal pathway is a major degradation pathway in cells that can degrade 80% of cellular proteins 26 . To confirm whether PAHwt, R241C, and R243Q proteins undergo protein degradation by the 26S proteasomal pathway, we transfected HEK293 cells with PAHwt, R241C, and R243Q variants and treated the cells with an increasing concentration of the MG132, a proteasomal inhibitor for 12 h. Our results indicate that the PAHwt and R241C variant proteins accumulated in a dosedependent manner as the MG132 concentration increased (Fig. 2a). However, the R243Q PAH protein level was about twofold lower than the PAHwt accumulated at 10 μM concentration of MG132 treatment, suggesting that the R243Q PAH variant is highly susceptible to proteasomal degradation (Fig. 2a).  www.nature.com/scientificreports/ In addition to the proteasomal system, recent reports suggested that the PAH protein undergoes degradation through the autophagy pathway 10,17,18 . Thus, we sought to determine the effect of increasing the concentration of lysosomal inhibitor ammonium chloride (NH 4 Cl) on PAHwt, R241C and R243Q proteins. Autophagy inhibition was confirmed by accumulation of the autophagosome marker LC3-II, which is an overall indicator of autophagy impairment. NH 4 Cl treatment increased protein levels of the PAHwt, R241C, and R243Q variants compared to untreated cells ( Supplementary Fig. S2), indicating that these variants might undergo degradation by an autophagy system. Since the main objective of this study was to analyze the deubiquitinating effect of USP19 on PAH variants R241C and R243Q, we expanded our research on UPS-mediated degradation of PAH variants.
Next, to investigate the ubiquitination of PAHwt and the mutant variants, HEK293 cells stably expressing PAHwt, R241C, and R243Q were transfected with Flag-tagged ubiquitin. Immunoprecipitation assays were then performed using PAH antibody, followed by Western blotting with specific endogenous antibodies against PAH and ubiquitin (Fig. 2b-e). Similarly, the ubiquitination of PAHwt and the mutant variants was cross-confirmed exogenously by co-transfecting HA-tagged PAHwt, R241C, R243Q, and Flag-ubiquitin. The cells were subjected to an immunoprecipitation assay using anti-HA antibody and immunoblotted with anti-Flag and anti-HA antibodies ( Supplementary Fig. S3). Our data show that the R241C and R243Q variants had higher ubiquitination smears than PAHwt (Fig. 2b-e and Supplementary Fig. S3). Interestingly, the R243Q variant showed an increase in conjugated ubiquitin molecules compared with the R241C variant (Fig. 2c,e), suggesting that the misfoldingrelated instability of the R243Q variant resulted in increased ubiquitination and is susceptible to the proteasomal degradative system. Protein band intensities were estimated using the ImageJ software with reference to the GAPDH control. The band intensity for HA-PAH/GAPDH was represented below the blots. (b-e) HEK293 cells stably expressing PAHwt, R241C, or R243Q were transfected with Flag-ubiquitin to evaluate their ubiquitination status. Cells treated with IgG and cells transfected with empty vector were used as the negative controls. (b, d) represent the total cell extract used in the immunoprecipitation (input); (c, e) represent immunoprecipitation using anti-PAH antibody and immunoblotted with anti-ubiquitin and anti-PAH (SE, short exposure; LE, long exposure). and R243Q, we transfected HEK293 cells with PAHwt, R241C, and R243Q along with USP19, co-immunoprecipitated them using anti-Flag and HA antibodies, and conducted immunoblotting with reciprocal antibodies. The anti-Flag antibody immunoprecipitated USP19 along with PAHwt and the R241C and R243Q proteins. In a reciprocal immunoprecipitation, anti-HA antibody immunoprecipitated PAHwt and the PAH variants along with USP19, indicating that USP19 interacts with PAHwt and the R241C and R243Q proteins (Fig. 3a). Our results showed that the interaction between USP19 and R243Q was weaker than that between USP19 and the R241C variant (Fig. 3a). One reason could be the lower R243Q protein abundance for interaction with USP19 because of its rapid proteolysis by the proteasomal degradative system. Additionally, we performed a Duolink PLA assay to analyze the interaction between USP19 and PAHwt and the PAH variants. As shown in Fig. 3b, in situ USP19 and PAH interaction was observed in the form of PLA dots when USP19 and PAHwt or the PAH variants were immunostained together (Fig. 3b, left panel), but not when they were stained with either USP19 or PAH antibody alone (Fig. 3b, right panel). Thus our data suggest that USP19 interacts with PAHwt and the PAH variants.

USP19 extends the half-life of PAH variant proteins through its deubiquitinating activity.
To examine the effect of USP19 on protein level of PAH variants, we transfected HEK293 cells with constant amounts of R241C and R243Q along with increasing concentrations of USP19 and analyzed by Western blotting. Our results demonstrate that USP19 offers dose-dependent increase in R241C and R243Q protein level (Fig. 4a). Next, we investigated the effect of USP19 on the protein turnover of the R241C and R243Q variants. To that end, we used the translation inhibitor CHX to examine the protein turnover from 0 to 8 h. We transfected HEK293 cells with R241C and R243Q proteins with and without USP19 and then treated the cells with CHX for 8 h. The half-lives of both R241C (Fig. 4b) and R243Q (Fig. 4c) were extended in the presence of USP19. Interestingly, the half-life of the highly unstable R243Q variant was significantly extended by USP19, suggesting that USP19 positively regulates the protein turnover of PAH variants. Next, we examined the effect of USP19 on the ubiquitination level of PAHwt and its variants. HEK293 cells stably expressing PAHwt, R241C, and R243Q were transfected with Flag-ubiquitin and Flag-USP19. PAHwt, R241C, and R243Q were then immunoprecipitated using PAH antibody, followed by immunoblotting using ubiquitin-and USP19-specific antibodies. Our results show that both of the PAH variants displayed increased polyubiquitination smears compared with PAHwt, which was reduced by the deubiquitinating activity of USP19 ( Fig. 5a,b, lane 5 and 6 vs. lane 7 and 8). To further validate those findings, we performed a Duolink PLA assay to determine the interaction between PAH and ubiquitin in the presence and absence of USP19. Our results demonstrate that USP19 overexpression reduced the interaction between ubiquitin and PAHwt and the PAH variants, compared with the mock control cells (Fig. 5c). Thus, USP19 has a key regulatory function on PAH variants by extending their half-lives, and preventing their degradation.
Next, we used the DUB inhibitor PR-619 and a previously validated sgRNA against USP19 from our CRISPRbased DUB knockout library 27 to transiently knock down USP19 levels, and analyzed the ubiquitination status of PAH. The efficiency of sgRNA targeting USP19 was analyzed on HEK293 cells stably expressing PAHwt by Western blot analysis. We observed that the protein levels of PAH were reduced when USP19 was depleted with . USP19 interacts with R241C and R243Q. (a) HEK293 cells were transfected with the indicated plasmids, followed by a co-immunoprecipitation assay to evaluate interactions between the PAH variants and USP19. Cells transfected with empty vector were used as the negative control. (b) HEK293 cells transfected with HA-tagged PAHwt, R241C, and R243Q were subjected to immunostaining using USP19 and PAH antibodies together (left panel). Cells stained with PAH or USP19 antibodies alone serve as negative control (right panel). DAPI was used to stain the cell nuclei, and the interaction was assessed using a Duolink PLA assay. Scale bar = 10 µm. Quantification of the PLA dots is shown as mean ± SEM.  . The band intensities of R241C and R243Q from (a-c) were estimated using ImageJ software, normalized to GAPDH, and represented graphically. Data are presented as the mean and standard deviation of three independent experiments. Šídák's multiple comparisons test was used to evaluate the P value (*P < 0.05, **P < 0.005, ****P < 0.00005; ns denotes non-significant). www.nature.com/scientificreports/ presence of USP19, the protein levels of R241C and R243Q variant were increased up to 1.9-fold and 1.3-fold, respectively (Fig. 6a). The functionality of PAHwt, R241C, and R243Q was tested by quantifying the Phe levels using HPLC in the presence of USP19. A standard curve was plotted using a known amount of Phe in triplicate and used to determine the Phe concentration in the samples (Fig. 6b). In the presence of USP19, the percentage of Phe in cells expressing R241C and R243Q was reduced by about 1.7-fold and 1.1-fold respectively (Fig. 6c,d).
In a reciprocal manner, we confirmed the amount of Tyr produced by R241C and R243Q using a tyrosine kit. A standard curve was plotted using a known amount of Tyr in triplicate and used to determine the Tyr concentration in the samples (Fig. 6e). Consistent with our HPLC results, the metabolism of Phe in cells expressing R241C and R243Q was increased by USP19. In the presence of USP19, the total amount of Tyr product in cells expressing R241C and R243Q was increased by about 1.6-fold and 1.3-fold, respectively (Fig. 6f). Overall, our results suggest that USP19 increases the cellular protein level of PAH variants by extending their half-lives, which results in increased Phe metabolism.

Discussion
PKU is a complex metabolic disorder caused by deficiency of PAH activity. Our understanding of PAH structure was recently advanced by the identification of the crystal structure for the full-length hPAH protein 22,23 .
The multidomain PAH protein exists in two distinct conformations, an auto-inhibited conformation and a distinct activated conformation. The transition from auto-inhibited structure to activated PAH conformation is dependent on Phe binding to an allosteric site of the N-terminal regulatory domain, causing large movements and dimerization of the regulatory domain exposing active sites 28 . With the rise in Phe level, equilibrium shifts from auto-inhibited to activated PAH conformation, maintaining Phe below a neurotoxic level 23,29 . More than 1,500 mutations have been identified in the PAH gene according to the locus-specific database, PAHvdb (http:// www. biopku. org/ pah/), among which over 500 mutations are associated with PKU 30-32 . Certain PKU-associated Data are presented as the mean ± SD of three independent experiments. One-way ANOVA followed by Tukey's multiple comparisons test was used (**P < 0.005; ns denotes non-significant). (d) The HPLC chromatogram of a PAH activity assay shows a Phe peak at a retention time of ~ 7.7 min. The Phe peak is indicated by a red arrow, and the measurement of the peak area is denoted at the top of the peak. (e) Standard curve of Tyr, representing an increasing concentration of Tyr in nmol, was plotted against the absorbance at OD 492 nm. (f) A tyrosine assay kit was used to analyze the residual activity of R241C and R243Q in the presence of USP19. Specific PAH activity is expressed as the amount of Tyr (nmol) produced by each sample. Data are presented as the mean ± SD of three independent experiments (**P < 0.005; ns, non-significant).  34 or to affect conformational stability (e.g. G46S, R408W, I65S) [34][35][36] . In addition, PKU-associated mutations might cause inability in the transition from auto-inhibited PAH to the activated conformation 29 . Therefore, diverse therapeutic approaches have to be investigated to address the breadth of PKU disease-causing mutants.
In this study, we considered PAH variants R241C and R243Q, which are prevalent in the Asian population and are associated with both mild and severe PKU, with residual activity of 25% and 12.8%, respectively 11 . A report suggested that high correlation exists between the structural destabilization of PAH variants and PKU disease severity 14 . Moreover, the misfolding-associated mutation in PAH acts as a trigger for rapid protein degradation, leading to insufficient availability of cellular PAH 10,18,37,38 . Likewise, we observed that the expression of both R241C and R243Q PAH variants was lower than that of the PAHwt protein. The R243Q variant in particular displayed about sixfold lower protein level than PAHwt (Fig. 1d), which was in line with previous reports 14,25 . Interestingly, we found that reduced levels of PAH variants were caused by ubiquitin-mediated proteasomal degradation, while treatment with proteasomal inhibitor was able to rescue PAH protein from degradation (Fig. 2).
The autophagy-lysosomal pathway is another major pathway that mediates the degradation of misfolded PAH variants. Cross-talk between UPS and autophagy pathways is critical for maintaining cellular proteostasis 18,39,40 . The PAH amyloid-like aggregates in PAH-R261Q mouse liver co-localize with autophagy markers such as P-p62 and LC3, which is a strong evidence of selective autophagy degradation of PAH-R261Q 18 . In addition to UPSmediated degradation of R241C and R243Q variants (Fig. 2a), we observed that dose-dependent treatment of lysosomal inhibitor gradually increased protein levels of R241C and R243Q variants ( Supplementary Fig. S2), suggesting that degradation of these variants is also associated with an autophagy pathway.
Chaperones and ubiquitin are important nodes mediating crosstalk between the proteasomal and autophagic degradation pathways. DNAJC12, a co-chaperone of HSP40, interacts with ubiquitinated PAH and might play an important role in HSP70-mediated folding or degradation of its client protein. Patients with biallelic mutations in the DNAJC12 gene were reported to exhibit HPA disorders with reduced PAH levels and activity 41 . The function of DNAJC12 was mutation-specific, where it increased the protein levels and enzymatic activity of a few mutant PAH variants such as p.L48S, p.I65T and p.R261Q, while reducing the protein levels and enzymatic activity of other PAH variants such as p.E280K, p.L348V, and p.V388M 41 . Moreover, increased cellular levels of PAH variants reduced disease severity as observed in the case of the F39L variant 42 . Therefore, we initiated this study to investigate whether USP19, a previously identified DUB for PAHwt 20 , can increase cellular levels of PAH variants through deubiquitinating activity.
Notably, USP19 is an endoplasmic reticulum (ER)-specific DUB and participates in the export of misfoldingassociated protein, which are associated with neurodegenerative diseases 43 . USP19 is frequently increased during the unfolded protein response and protects ER-associated degradation of substrates such as CFTR ΔF508 from proteasomal degradation through its deubiquitinating activity 44 . Interestingly, USP19 is the first DUB found to have activity that is promoted by HSP90, a chaperone protein. The N-terminal region of USP19 contains two CS/ P23 domains and interacts with HSP90 and enhances substrate recognition 45,46 . The USP19-HSP90 machinery has been proposed to be involved in regulating the stability of huntingtin and ataxin-3 proteins, playing a crucial role in the protein triage decision associated with the aggregation and protein degradation 46 . In contrast, it is possible that DUBs enhance the refolding efficacy of chaperone and thus cooperate with chaperones and promote stabilization of their target substrate. Previously, we demonstrated that USP19 extends the half-life of PAHwt protein by preventing its proteasomal degradation 20 . In the current study, we investigated the role of USP19 on variant PAH protein turnover and its metabolic function.
We observed that USP19 positively regulates and binds with the R241C and R243Q PAH variants similar to the PAHwt protein (Fig. 3a). Additionally, an in situ Duolink PLA assay also showed interaction between USP19 and R241C (Fig. 3). However, the R243Q variant, which is associated with severe PKU, showed relatively less interaction with USP19 (Fig. 3a,b), possibly due to the lower abundance of R243Q protein levels in the cell. Next, we showed that the R243Q variant undergoes accelerated degradation, with a shorter half-life than the R241C variant (Fig. 4b,c). To support that finding, we demonstrated that R243Q was highly ubiquitinated compared with both R241C and PAHwt (Figs. 2, 5) and hence displayed lower protein levels. Deubiquitinating enzymes can specifically counteract the ubiquitination of their substrate and have therapeutic value by regulating protein stability, aggregation, and degradation in diseases such as cystic fibrosis, Huntington's disease, and Parkinson's disease [47][48][49][50][51] . Here, we demonstrated that USP19 deubiquitinated and restored sufficient amounts of PAH variant enzymes in cells by extending the half-lives of both R241C and R243Q (Figs. 4, 5). In contrast, on transient knockdown of USP19, both PAH variants were rapidly degraded, demonstrating the specificity of USP19's deubiquitinating activity on PAH variants (Fig. 5d). Because these disease-linked PAH variants have shorter half-lives than PAHwt, an insufficient amount of cellular PAH protein is available, increasing disease severity 14 . Therefore, we investigated the functional consequence of USP19-mediated increase in the protein levels of the PAH variants on their metabolic function. Our results indicate that USP19 promotes Phe metabolism, which was quantified by findings of reduced Phe and increased product Tyr in HPLC and colorimetric assays (Fig. 6). The Phe metabolism exhibited by R241C was improved by USP19, but USP19 had only a subtle effect on protein level and Phe metabolism of the R243Q variant (Fig. 6), suggesting that the function of USP19 is dependent on the variant. Altogether, to develop better therapies for PKU-disease, it is necessary to further investigate the regulatory mechanisms of USP19 alone or in combination with chaperones to elevate cellular protein levels in PAH variants such as F39l, P122Q, F161S, and I65T, which are all susceptible to rapid proteasomal degradation 14

Stable expression of PAH and variants in HEK293 cells. To generate HEK293 cells that stably
expressing PAHwt, R241C, and R243Q, we used the pLVX-IRES-ZsGreen1 plasmid encoding PAHwt, R241C and R243Q for lentiviral production. One day prior to transfection, 1 × 10 6 HEK293 cells were seeded and cultured in a 100 mm culture dish. The HEK293 cells were then co-transfected with pLVX-IRES-ZsGreen1 plasmids encoding PAHwt, R241C, or R243Q and packaging vectors (pLP1, pLP2 and pLP-VSVG) in a 4:1:1:1 ratio. Cell supernatants were harvested 48 h after transfection and either used to infect cells or stored at − 80 °C. Cells were infected for 6 h with the lentiviral supernatants diluted 1:1 with normal culture medium in the presence of 10 μg/mL of polybrene (Sigma-Aldrich) to obtain stable HEK293 cell lines expressing PAHwt or a PAH variant. The transduction efficiency of pLVX-ZsGreen1 plasmid encoding PAHwt, R241C and R243Q was checked after 72 h (Supplementary Fig. S1a and S1b). The cells transduced with empty vector were used as control.
Immunoprecipitation. HEK293 cells were lysed with IP lysis buffer containing 150 mM sodium chloride, www.nature.com/scientificreports/ ously described 53 . Briefly, 120 μg of total protein was pre-incubated with 1 mmol/L of l-Phe (P17008, Sigma, St. Louis, MO, USA) and 2 µg of catalase (C1345, Sigma) in 0.1 mol/L Na-HEPES buffer (pH 7.0, T&I, BHE-9000, Gangwon, Korea) for 5 min followed by a 1 min incubation with 10 mM ferrous ammonium sulfate. The reaction was initiated by adding 200 µmol/L of BH 4 (Cat no #T4425, Sigma) in 5 mM DTT (Cat no. #10197777001, Sigma) and allowed to proceed for 30 min at 25 °C. The reaction was stopped by adding 50 µL of 2% (w/v) acetic acid in ethanol. All concentrations mentioned refer to the final concentration in a 100 µL reaction mixture. The amounts of Tyr were determined using a tyrosine assay kit (Cat no. #ab185435) according to the manufacturer's protocol. A standard curve for Tyr was used to determine the Tyr concentration in the samples (Fig. 6e).
For the HPLC-UV analysis, a Phenomenex EZ:Faast™ kit was used to prepare the samples. The amount of Phe was determined using an HPLC system with a UV detector at a wavelength of 210 nm. Acetonitrile (94:6 v/v), 20 mmol/L sodium acetate buffer (pH 6.5), and a Shiseido Capcell Pak MF C 8 analytical column (4.6 mm × 150 mm) were used for the chromatography. The flow rate was 1 mL/min, and the sample injection volume was 20 µL. A standard curve for Phe was used to determine the Phe concentration in the samples (Fig. 6b). The chromatograms represent the peak height (in arbitrary units, AUV) against the retention time (Fig. 6d).
Statistics. The statistical analysis was conducted using GraphPad Prism 9 (GraphPad Software, Inc. San Diego, CA, USA), and the results are presented as the mean ± standard deviation of three independent experiments. One-way ANOVA and paired t testing were used to analyze the data, and multiple comparisons among groups were performed with Tukey's post hoc test or Šídák's multiple comparisons test. For comparisons between two groups, two-way ANOVA was used to analyze the data (*P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.00005, and ns denotes non-significant).